Ribosome-inactivating glycoproteins, modified by oxidation of their osidic units and formation of a schiff&#39;s base and in-vivo prolonged action immunotoxins containing such a glycoprotein

ABSTRACT

Glycoprotein which inactivates ribosomes (GPIR) the ribosome-inhibiting activity of the native GPIR and having a prolonged-action in vivo which is obtained by oxidation of its osidic units by the action of periodate ions, and simultaneous blocking of the oxidation product by formation of a Schiff&#39;s base with a suitable primary amine. Said modified glycoprotein may be coupled to an antibody or a fragment thereof in order to form an immunotoxin.

This application is a division of application Ser. No. 06/941,990 filedDec. 15, 1986, now U.S. Pat. No. 4,911,911.

The present invention relates to new medicinal molecules containing atleast one antibody covalently bonded to a constituent of polypeptidetype which inhibits protein synthesis and is derived from a glycoprotein(or a glycopeptide) whose polysaccharide units have been modified.

U.S. Pat. No. 4,340,535 and French Patent Applications published underNo. 2 504 010 and 2 516 794 describe the preparation of anticancerproducts, called conjugates, which are obtained by the coupling, bymeans of a covalent bond, of the A chain of ricin with antibodies orantibody fragments directed against antigens carried by the cell to bedestroyed. The products of this type have been designated, and aredesignated in the present Application, by the generic name ofimmunotoxins.

Conjugates analogous to the previously described immunotoxins containingthe A chain of ricin are known which are also suitable as anticancerdrugs and result from the coupling, by means of a covalent bond, ofantibodies or antibody fragments with other glycoproteins whichinactivate ribosomes, such as, in particular, the gelonine extractedfrom Gelonium multiflorum (Eur. J. Biochem. 1981, 116, 447-454; CancerRes. 1984, 44, 129-133) or the inhibitor extracted from Momordicacharantia (MOM) (U.S. Pat. No. 4,368,149).

These glycoproteins which inactivate ribosomes (abbreviated to GPIR),and which have properties similar to those of the A chain of ricin, aresubstances with a molecular weight of the order of magnitude of 20,000and 30,000 (Cancer Survey, 1982, 1, 489-520).

The term "glycoprotein which inactivates ribosomes", as used in thepresent description and also in the claims, denotes any substance whichcarries saccharide units belonging to the class of macromolecules whichinactivate ribosomes and consequently inhibit protein synthesis ineucaryotic cells, as well as any fragment of the said substance whichpossesses the same inactivating property, it being possible for the saidglycoprotein which inactivates ribosomes to be of natural orbiosynthetic origin, being derived from a cell whose genotype has beenmodified for this purpose.

It is also known that the cytotoxic activity of these immunotoxins canbe potentiated by a variety of adjuvant substances such as ammoniumsalts, various amines or certain carboxylic ionophores such as monensinor nigericin.

However, the therapeutic effects of immunotoxins, whether activated ornot, can only manifest themselves fully on condition that theimmunotoxin is capable, through its antibody part, of becoming localizedin vivo, in the active form, on the target cells to be destroyed (sinequa non condition for any expression of immunotoxin activity). Thecapacity of the immunotoxin to become localized on the target dependsfirst and foremost on the ability of the immunotoxin to remain in thebloodstream and the extracellular fluids, in the active form, forsufficient lengths of time for it to reach its target cells and atsufficient concentrations to give a high degree of occupation of thecorresponding antigen sites.

Numerous studies have made it possible to establish the plasmaelimination kinetics of immunotoxins after intravenous injection intovarious animal models. It has been found that, after injection, theplasma level of biologically active immunotoxin decreases very rapidlyand very substantially. Thus, in a typical case involving rabbits, in amodel using an immunotoxin built up by coupling the A chain or ricin, bymeans of a link containing a disulfide bridge, with a monoclonalantibody directed against the antigen T65 of human T lymphocytes(antibody T101), it is found that 97% of the immunotoxin present in thebloodstream at time 0 after injection disappears in 30 minutes and 99.9%disappears in 17 hours. This rapid disappearance of the immunotoxinquite obviously detracts from the expression of its complete cytotoxiccapacity, the immunotoxin being prevented from durably saturating a highproportion of the target antigens carried by the cells to be destroyed.Moreover, a comparison of the plasma elimination kinetics ofimmunotoxins with those of the corresponding unconjugated antibodiesslows by contrast that--as is well known--the antibodies remain in theplasma at a high level for relatively long periods of time. Now, even inthe most highly purified immunotoxin preparations, there is always acertain residual level of unconjugated antibodies. Due to the effect ofthe differential rates of elimination of immunotoxins and antibodies,the unconjugated antibodies, which are initially very much in theminority, progressively become the majority component after a few hours,so these antibodies gradually compete to become powerful antagonists forthe fixation of the immunotoxins to their targets.

Therefore, this clearly shows the value of enhancing the persistence ofimmunotoxins in the plasma, in their active form, so as to increase boththe duration and degree of occupation of the target antigens andconsequently to improve the therapeutic effects of the immunotoxins.

Furthermore, experiments involving in vivo localization of theimmunotoxin containing the A chain of ricin, radiolabeled and theninjected into animals with no specific target, have shown that theconjugate becomes localized preferentially in the liver during the firstfew minutes after injection. The same applies to the A chain of ricin,which follows the same pattern when injected in the uncoupled form. Thisstrongly suggests that the immunotoxin becomes fixed in the liver viathe cytotoxic sub-unit contained in the immunotoxin.

It is known that the A chain of ricin is a glycoprotein whose polyosidicgroups comprise especially mannose residues and N-acetylglucosamineresidues, some mannose residues being in terminal positions (Agri. Biol.Chem., 1978, 42, 501). Also, receptors capable of recognizingglycoproteins containing these terminal mannose residues have been foundto exist in the liver. It has also been shown that the glycoproteinsrecognized by these receptors--the latter being present essentially onthe Kupffer cells--are rapidly eliminated from the bloodstream byfixation to these cells, which metabolize them. This is well documentedespecially in the case of beta-glucuronidase and in the case ofribonuclease B (Arch. Biochem. Biophys., 1978, 188, 418; Advances inEnzymology, published by A. Meister, New York, 1974; Pediat. Res., 1977,11, 816).

Taken as a whole, this information shows that the rapid elimination ofimmunotoxins containing the A chain of ricin can be explained by therecognition of the mannose residues of the A chain of ricin by thehepatic cells and in particular the Kupffer cells.

The studies of plasma elimination kinetics carried out on other GPIRs,for example gelonine or MOM, after intravenous injection into theanimal, have shown that, as in the case of the A chain of ricin, theplasma level of GPIR decreases very rapidly and very substantially afterinjection. Thus, in a typical case involving rabbits, after theinjection of gelonine purified by the method described (J. Biol. Chem.,1980, 255, 6947-6953), it is found that 93% of the gelonine present inthe bloodstream at time 0 after injection disappears in 1 hour and99.99% disappears in 24 hours.

It is known that the oxidation of osidic structures, including thosecontained in glycoproteins, with periodate ions causes the scission ofthe carbon chain wherever two adjacent carbon atoms carry primary orsecondary hydroxyls. If the two adjacent hydroxyls are secondary, as isgenerally the case in the cyclic oses present in GPIRs, oxidationproduces two aldehyde groups on the carbons between which the scissionhas taken place.

It is also known that the aldehyde groups are very active towards theprimary amine groups by formation of imines also known as Schiff'sbases. Thus, the aldehyde groups formed during the oxidation reactioncan react with the primary amines carried by the peptide chain ofglycoprotein, and form undesirable intra- and/or intermolecular covalentbonds, leading to an instability of the oxidation product and often tothe formation of insoluble polymers.

It has now been found, absolutely unexpectedly, that, if thecarbohydrate units of a GPIR are modified by the original processdescribed hereinafter, a new molecule of GPIR is obtained which has thedual property of retaining its biological activities and of beingeliminated very slowly from the bloodstream after injection to superioranimals or humans. This new modified GPIR which retains the property ofinactivating ribosomes and which, due to the modification, has acquireda long period of action in vivo, is designated in the presentapplication by the symbol GPIR-1a.

Said original process consists in modifying the osidic units of the GPIRby reaction with periodate ions in the presence of an excess of anauxiliary reagent comprising a primary amine group and which, by therest of the structure, is not likely to react with the periodate ions inthe operational conditions used. Thus, the aldehyde groups created bythe oxidation with periodate are blocked as and when they appear byformation of Schiff's base with the excess of exogene amino reagent,thus avoiding undesirable reactions with other amino groups of the GPIRand permitting the obtention of a stable and perfectly soluble product.

It has also been found that if these new prolonged-action glycoproteinsare coupled with antibodies or antibody fragments, the resultingconjugates retain the known biological properties of immunotoxins andexhibit slow plasma elimination kinetics.

The present invention therefore relates, as new product, to astructure-modified GPIR, whose carbohydrate units have been modified bythe action of periodate ions in the presence of an excess of auxiliaryreagent presenting a primary amine group and capable of blocking thealdehyde groups created by the oxidation with periodate, as and whenthese appear. The invention further relates to the process for obtainingsaid modified GPIR such as described hereinabove.

The present invention further relates to products, belonging to theclass of the immunotoxins, which are obtained by the covalent couplingof, on the one hand, an antibody or antibody fragment, used in itsnatural form or correctly modified, and, on the other hand, a moleculeof GPIR whose carbohydrate units have been modified by the aforesaidoriginal process.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the plasma elimination curves as a function of time fornative A chain (curve 1) and a modified A chain (curve 2) according tothe present invention.

FIG. 2 shows the plasma elimination curves as a function of time for animmunotoxin (curve 1) and a modified immunotoxin conjugate (curve 2)according to the present invention.

The meaning is given hereunder of the different products used incarrying out the invention.

The term "periodate" denotes the IO₄ ⁻ ion present in aqueous solutionsof periodic acid salts and in particular salts deriving from alkalinemetals. Said salts are also mentioned in the literature under the nameof metaperiodates.

The term "auxiliary reagent having a primary amine group" denotes anyorganic molecule carrying a single primary amine group which is notlikely to react with the periodate ions in the operational conditionsused. For example, said reagent may be any alkylamine, non reacting withthe periodate, and soluble in water, at the necessary concentration, inbase or salt form, at a pH varying between 5 and 7, and at a temperaturevarying between 0° and 15° C. Said reagent may also be any α-amino orω-amino acid meeting the aforesaid conditions, whether it is opticallyactive or racemic, such as one of the following aliphatic amino acids:glycine, alanine, valine, leucine, isoleucine, arginine, aspartic acid,glutamic acid. Said reagent may also be any peptide meeting theaforesaid conditions.

The term "antibody" denotes a protein selected from any antibody orantibody fragment, or any immunoglobulin or immunoglobulin fragment orany molecule derived from the above by artificial modification of anyone of their functional groups, including osidic structures that theycarry, with the proviso that the protein chosen in this way remainscapable of selectively recognizing a given antigen on the surface of thecells carrying this antigen, and in particular the target cells. Thestarting antibody may be of natural or biosynthetic origin, beingderived from a cell whose genotype has been modified for this purpose.

The preparation of monoclonal antibodies directed in particular againstdefinite human target cells has been widely covered in the scientificliterature and many of these antibodies are now available on the market.

The symbol P represents a protein chosen from the group comprising anyantibody or antibody fragment, any immunoglobulin or immunoglobulinfragment or any molecule derived from the above by artificialmodification of any one of their functional groups, includingcarbohydrate structures which they carry, with the proviso that theprotein chosen in this way remains capable of selectively recognizing agiven antigen on the surface of the cells carrying this antigen,especially the cancer cells. The starting protein P can be of natural orbiosynthetic origin, being derived from a cell whose genotype has beenmodified for this purpose.

The symbol GPIR represents a glycoprotein or one of its fragments.Provided that such fragments retain all or part of theribosomes-inactivating property which characterizes the GPIR from whichthey are issued, they can be used as starting products, but the nativeGPIR is preferred.

The symbol "GPIR-1a" represents the GPIR modified according to theinvention, namely a molecule having the property of inactivating theribosomes like the GPIR but having a period of action in vivo which ishigher than that of the GPIR and which results from the treatment inaqueous medium of the GPIR by an oxidizing agent, such as periodate, inthe presence of an excess of primary amine in order to transform theformed aldehyde groups into imine groups (or Schiff's base).

The operation is in general conducted at a temperature varying between0° and 15° C., in an aqueous solution of pH between 5 and 7, andpreferably in the absence of light; in this condition, the reactionlasts between 0.2 and 24 hours.

In the immunotoxin, the GPIR-1a part is also denoted as a "cytotoxicsub-unit".

The symbol A--1a represents a prolonged-action glycoprotein whichinactivates ribosomes, obtained by treatment of the A chain of ricin, inwhich at least one of the thiol groups of its cysteines 171 and 257 isoptionally protected, with an aqueous solution of an alkali metalperiodate, for a period of 0.2 to 24 hours, at a temperature of 0° to15° C. and in the absence of light, and by deprotection of the saidthiol group, if appropriate.

The symbol P' represents a radical derived from the above protein P, assuch as appropriately chemically modified, from which one or more of itsown groups have been removed and in which other functional groups areoptionally blocked.

The symbol GPIR-1a' represents a radical derived from the above proteinGPIR-1a, as such or appropriately chemically modified, from which one ormore of its own groups have been removed and in which other functionalgroups are optionally blocked.

The symbol A--1a' represents a radical derived from the protein A--1a,from which at least one of the thiol groups of its cysteines 171 and 257have been removed.

The symbol P₁ represents one of the proteins GPIR-1a and P as definedabove, which carries free thiol groups attached to the said proteindirectly or via a spacing structure.

The symbol P₂, which is different from P₁, represents one of theproteins GPIR-1a and P as defined above, which carries one or morefunctional groups capable of reacting with the free thiols.

The symbol P₁ ' represents that radical of the protein P₁ which isbonded to the functions belonging to the protein P₁, especially thegroups SH (of the cysteine), NH₂ (in the terminal position of theprotein or in the epsilon position of the lysines), OH (of thetyrosines) of COOH (of the aspartic and glutamic acids), or, only in thecase where P, is an antibody or antibody fragment, that radical of theprotein P₁ which originates from the opening of the carbohydratestructures by reaction with periodic acid according to known methods.

The symbol P₂, represents that radical of the protein P₂ which is bondedto the characteristic functional groups NH₂ (in the terminal position ofthe protein or in the epsilon position of the lysines), OH (of thetyrosines) or COOH (of the aspartic and glutamic acids).

For example, P_(1') --SH represents the protein P₁ (which canarbitrarily be the antibody or antibody fragment P or the proteinGPIR-1a) in which the SH groups of the cysteines are free and the otherfunctional groups are optionally blocked.

In the same way, P_(1') --CO-- represents the protein P₁ in which theterminal carboxyl group or the carboxyl groups of its glutamic andaspartic acids are coupled with a group which artificially introduces anSH group.

Again, P_(2') --NH-- represents the protein P₂ (which can arbitrarily bethe antibody or antibody fragment P or the protein GPIR-1a) in which theterminal amino group or the amino groups of its lysines are attached toa group capable of coupling with the thiol of the protein P₁.

The term "inert spacing structure", as used here for E and E', denotes adivalent organic radical which in inert towards the reactants used inthe process, such as a straight-chain or branched alkylene groupcontaining from 1 to 15 carbon atoms, which can obtain one or moredouble bonds, can be interrupted by oxygen atoms or can be substitutedby one or more inert functional groups such as methoxy groups, free oresterified carboxyl groups, dialkylamino groups or carbamate groups. Thesame term also denotes an arylene group containing from 6 to 15 carbonatoms, which can be substituted by one or more inert functional groupsas indicated above for the alkylene group.

The expression "functional group capable of bonding covalently", as usedhere for Y and Y', denotes any groups capable of reacting with thegroups belonging to the proteins P₁ and P₂ to give a covalent bond.Thus, the groups --CO-- and --(C═NH)-- are suitable functional groupscapable of bonding with the free amines, the thiols and the phenolichydroxyls of the proteins. Likewise, the --NH-- group is a suitablefunctional group capable of bonding with the free carboxyl groups of theproteins. The group ═N-- is a suitable functional group capable ofbonding with the two carbon atoms of the carbohydrate structures of theproteins P₁ and P₂ after oxidation with periodate ions, but only in thecase where P₁ and P₂ are an antibody or an antibody fragment.

The expression "group belonging to the proteins", as used here for Z andZ', denotes the radicals originating from the characteristic groups ofthe amino acids forming the proteins P₁ and P₂, such as the oxygen atomoriginating from the hydroxyls of the tyrosine and possibly serine aminoacids, the carbonyl group originating from the terminal carboxyl or thefree carboxyls of the aspartic and glutamic acids, the --NH-- grouporiginating from the terminal amine of the proteins, for example thelysine, or the sulfur atom originating from the thiol of the cysteine.The same expression also denotes the group originating from thedialdehyde structure obtained after oxidation of one of the carbohydratestructures of the proteins P₁ and P₂ by treatment with periodate ions,but only in the case where P₁ and P₂ are an antibody or antibodyfragment.

The term "activating radical", as used here for X, denotes a groupbonded to an --S--S-- bridge and capable of reacting with a free thiolto form a disulfide with the release of X-SH. Suitable activatingradicals are pyridin-2-yl and pyridin-4-yl groups which areunsubstituted or substituted by one or more halogens or alkyl, carboxylor alkoxycarbonyl radicals; the phenyl group which is unsubstituted or,preferably, substituted by one or more halogens or nitro, alkoxy,carboxyl or alkoxycarbonyl groups; or an alkoxycarbonyl group such asmethoxycarbonyl.

The terms "alkyl" and "alkoxy" denote groups containing up to 5 carbonatoms.

The term "alkylene" denotes straight-chain or branched saturatedaliphatic groups containing up to 10 carbon atoms, which can besubstituted by one or more inert functional groups such asalkoxycarbonyl groups.

Glycoproteins which inactivate ribosomes and which are used as preferredstarting materials for oxidation with periodate ions, and for reductionaccording to the invention, are all GPIRs, such as the A chain of ricin,which are in themselves only very slightly cytotoxic because they cannotfix to the cells, but which, on the other hand, after coupling with anantibody recognizing particular cells, become highly cytotoxic towardsthese cells once the antibody has recognized its target.

Representative starting compounds are the A chain of ricin, gelonine andthe substance extracted from Momordica charantia (MOM), as obtained byextraction.

Other GPIRs which are useful as starting materials for oxidation withperiodate ions are as follows:

    ______________________________________                                        Dianthin 30      from Dianthus caryophyllus                                   Dianthin 32      from Dianthus caryophyllus                                   Agrostin A       from Agrostemma gitnago                                      Agrostin B       from Agrostemma gitnago                                      Agrostin C       from Agrostemma gitnago                                      HCI              from Hura crepitans                                          Asparagus officinalis                                                                          from Asparagus officinalis                                   inhibitor                                                                     ______________________________________                                    

The same substances produced biosynthetically by cells whose genotypehas been modified for this purpose are also suitable compounds.

Fragments of the above GPIRs, provided they retain all or part of theproperty of inactivating ribosomes which characterizes the GPIR fromwhich they are derived, can also be used as starting materials.

The native A chain of ricin in which at least one of the thiol groups isprotected is a preferred starting compound.

The preparation of the pure A chain of ricin is described in U.S. Pat.No. 4,340,535. Gelonine and MOM are also described.

Protection of the thiol groups of the starting GPIRs is only necessaryif the said thiol groups are those which are to used for coupling withthe antibody. If other functional groups are used for the coupling, forexample the phenolic hydroxyl of the tyrosines, protection is notcarried out.

Blocking is carried out by reaction with a reagent capable ofsubstituting the SH groups with a radical which can subsequently beremoved by reduction or thiol/disulfide exchange, for example2,2'-dinitro-5,5'-dithiodibenzoic acid (DTNB) or alternatively3-(pyridin-2-yldisulfanyl)propionic acid. In the absence of such atreatment, the free thiols of the A chain may disappear during theoxidation reaction, in which case they cannot be totally regenerated byreaction with a reducing agent such as 2-mercaptoethanol. The excessblocking agent is removed by dialysis or any other appropriatetreatment.

The GPIR whose thiols are optionally blocked is then subjected to theoxidation reaction with the periodate ions and to the simultaneousformation of Schiff's base with a primary amine.

The periodic oxidation reaction and the blocking reaction by formationof Schiff's base may be carried out at a moderately acid pH, such asbetween 5 and 7 and preferably between 6 and 6.5. The GPIR is mixed withthe primary amine before the periodate is added. The periodate is usedin excess; more particularly, the concentration of alkali metalperiodate is always greater than the concentration of vicinal diolscapable of being oxidized: concentrations of 10 to 50 mM in respect ofsodium periodate for concentration of 1 to 10 mg/ml of cytotoxicsub-unit are suitable. The primary amine (such as L-leucine, L-alanine,L-arginine or L-glutamic acid) is also used at a concentration which isgreater than the concentration of vicinal diols capable of beingoxidized: concentration of 50 to 500 mM of primary amine forconcentration of 1 to 10 mg/ml of cytotoxic sub-unit are suitable. Thetreatment, carried out at a temperature between 0° and 15° C. andpreferably between 1° and 5° C. and in the dark, takes between 0.2 and24 hours.

When the reaction is over, the excess of periodate is eliminated byadding a reagent which consumes it, such as for example, an excess ofethylene-glycol, and the by-products are removed by dialysis. Theproduct obtained at the end of the reaction is isolated by theconventional techniques.

If the thiol groups of the starting material have been blocked,unblocking is effected by the known methods, for example by reactionwith a reducing agent capable of freeing the previously blocked thiolgroup, such as 2-mercaptoethanol, giving the new prolonged-actionglycoprotein which inactivates ribosomes, ready to be used for couplingwith an antibody to give an immunotoxin.

In the case of the A chain of ricin, the new molecule obtained in thisway (referred to by the symbols A--1a)possesses the following mainproperties:

a molecular weight which is not significantly different from that of thenative A chain. As far as it is possible to see by polyacrylamidegradient electrophoresis, this modification only produces polymers ofthe protein in a very small quantity and does not produce anydegradation products.

a proportion of free thiol groups greater than 0.7 per mol.

an immunoreactivity towards rabbit antibodies directed against the Achain of ricin which is indistinguishable from that of the native Achain.

an inhibitory activity on a protein synthesis in an acellular modelwhich is greater than 50% of that caused by an equal quantity of nativeA chain.

finally, after a single intravenous administration to rabbits at a doseof about 0.4 mg/kg of body weight, the plasma level of theprolonged-action A chain (A--1a) present in the bloodstream 23 hoursafter injection is 10 to 100 times greater than the plasma level of thenative A chain measured in the same conditions.

A GPIR prepared as described hereinabove can be used for preparingconjugates or immunotoxins according to the heretofore known methods.

More particularly, the present invention relates to products, belongingto the class of the immunotoxins, (hereinafter named IT) which areobtained by the covalent coupling of, on the other hand, an antibody orantibody fragment, use in its natural form or correctly modified, whichpossesses the capacity to selectively recognize an antigen carried bythe intended target cells, with, on the other hand, a prolonged-actionglycoprotein GPIR which inactivates ribosomes named GPIR-1a, obtained bythe process hereinabove disclosed, the coupling of the 2 proteins beingeffected either via a disulfide bond or via a thioether bond.

An immunotoxin formed by the coupling of an antibody P with aprolonged-action glycoprotein which inactivates ribosomes, GPIR--1a, canbe represented by the following statistical formula:

    P'--W--GPIR--1a'                                           (I)

in which P' represents the radical of a protein which is an antibody oran antibody fragment P, as such or appropriately chemically modified, inwhich other functional groups are optionally blocked, GPIR--1a'represents the radical of a protein which is GPIR--1a, as such orappropriately chemically modified, in which other functional groups areoptionally blocked, and W represents a divalent covalent structurecontaining a thioether group or a disulfide group in which either thesulfur atoms are those of the cysteines of P and GPIR--1a or they arebonded to the groups belonging to P and/or GPIR--1a by spacingstructures carrying a functional group bonded to the said groupsbelonging to P and/or GPIR--1a.

A thioether bond between two proteins is understood as meaning a bond ofthe type: ##STR1## in which Z, Y and E are as defined below.

The present invention preferentially relates to an imunotoxin of thestatistical formula:

    P'--W'--GPIR--1a'                                          (II)

in which P' and GPIR--1a' are as defined and W' represents a covalentstructure chosen from:

(a) a group of the formula: ##STR2##

(b) a group of the formula: ##STR3##

(c) a group of the formula

    --Z--Y--E--S--S--(E'--Y'--Z').sub.n --

or

(d) a group of the formula:

    --(Z'--Y'--E').sub.n --S--S--E--Y--Z--,

in which:

Z and Z', being either identical or different represent the groupsbelonging to the proteins GPIR--1a and P, chosen from the oxygen atomoriginating from the hydroxyl of one of the tyrosine residues, thecarbonyl group originating from one of the terminal carboxyls or thefree carboxyls of the aspartic and/or glutamic acids of GPIR--1a and P,the --NH-- group originating from one of the terminal amines of GPIR--1aand P or from one of the amines in the epsilon position of one of thelysine residues, and, only for Z in the covalent structures (b) and (c),the group originating from the dialdehyde structure obtained afteroxidation of one of the carbohydrate structures of P with periodic acidaccording to the known methods;

Y and Y' represent functional groups capable of bonding covalently withany one of the groups Z and Z' of the proteins GPIR--1a and P;

E and E' represent inert spacing structures; and

n represents zero or 1.

The immunotoxin of the present invention are represented in simplifiedform by the formula I and II above, but it is understood that there canbe several structures --W-- or --W'-- bonded to one and the samemolecule P and/or of molecule GPIR--1a, hence several GPIR--1a bonded toonly one P and vice-versa; the number of bonds depending on the couplingmethod and on the number of groups belonging to P and to GPIR--1a. Thus,statistical formulae I and II also represent these products and theirmixtures, of formula:

    P'(W'--GPIR--1a').sub.m

in which m is an integer or a mixed number, smaller or greater than 1.

For example, if an immunotoxin is formed by the coupling of the sub-unitA of native ricin with the antibody P (for example the antibody T101)via a divalent covalent structure having a disulfide group in which onesulfur is that belonging to the cysteine 257 of the prolonged-action Achain of ricin and the other is bonded to the phenolic oxygens of thetyrosines of the antibody P by an oxopropyl group, it will have thestatistical formula:

    P'(O--CO--CH.sub.2 --CH.sub.2 --S--S--A--1a').sub.t

in which t represents the number of tyrosines in the antibody (forexample the antibody T101) which are involved in the coupling.

The resulting immunotoxin thus corresponds to a product of the formulaII in which:

P' is as defined above, especially the radical of the antibody T101 fromwhich the phenolic groups of the tyrosines involved in the coupling havebeen removed;

A--1a' is the radical of the prolonged-action A chain of ricin fromwhich the thiol group of its cysteine 257 has been removed; and

W' is the group (c):

    --Z--Y--E--S--S--(E'--Y'--Z').sub.n --

in which Z is the oxygen of the phenolic hydroxyls involved in thecoupling, Y is --CO--, E is the inert spacing structure --CH₂ --CH₂ --and n is zero.

Particular preference is given to the immunotoxins formed by one or morestructures containing the prolonged-action sub-unit A of ricin and asingle antibody P, which are represented by the statistical formula:

    P'(W'--A--1a').sub.m                                       III

in which P', W' and A--1a' are as defined above and m represents thenumber of groups belonging to the protein P which are involved in thecoupling. The number m varies from 0.3 to 12, preferably from 0.5 to 10.

The expression "m varies from 0.3 to 12, preferably from 0.5 to 10"means that the value of m is a statistical value because the couplingdoes not take place homogeneously within the population of antibodymolecules. The number m may therefore not be an integer.

The value of m depends especially on the antibodies used and moreparticularly on their molecular weight.

Thus, if a fragment Fab or Fab' is used as the starting antibody P, thevalue of m can vary between 0.3 and about 2; if a fragment F(ab')₂ isused, m can vary between 0.5 and about 4; for an antibody of the IgGtype, the value of m will be between 0.5 and about 6; finally, for anantibody IgM, the value of m can vary between 1 and about 12.

It is preferable, however, for the degree of substitution on theantibody P to be such as to lead to a value of m which is not less than0.5 and not more than 10.

More generally, the structures I and II above represent statisticalformulae written in simplified form, as explained above.

Analogously, the formulae IV, V and IX below are also statisticalformulae--whenever is 1--because the coupling reactants are preparedfrom populations of proteins P₁ and P₂ which all have exactly the sameproperties as those considered above for the antibody P, whether theseproteins P₁ and P₂ are themselves the antibody P or the proteinGPIR--1a.

According to another feature, the present invention relates to a processfor the preparation of a prolonged-action immunotoxin having a covalentbond of the disulfide or thioether type between an antibody and aglycoprotein which inactivates ribosomes, wherein a disulfide orthioether bond is formed between an antibody and a prolonged-actionglycoprotein which inactivates ribosomes, obtained by treatment of aglycoprotein which inactivates ribosomes, the thiol groups of which areoptionally protected, with an aqueous solution of an alkali metalperiodate, for a period of 0.2 to 24 hours, at a temperature of 0° to15° C. and in the absence of light, in the presence of a primary amine,and by unblocking of the thiol group, if appropriate.

According to a preferred feature, the present invention relates to aprocess for the preparation of an immunotoxin having the structure Iabove, wherein a protein P₁, which is arbitrarily either theprolonged-action glycoprotein which inactivates ribosomes, GPIR--1a, oran antibody or antibody fragment, carrying at least one free thiol groupattached to the said protein P₁ directly or via a spacing structure, isreacted, in aqueous solution and at room temperature, with a protein P₂,which is different from P₁ and is arbitrarily either theprolonged-action glycoprotein which inactivates ribosomes, GPIR--1a, oran antibody or antibody fragment, carrying a group capable of couplingwith the free thiol of the protein P₁, so as to form a thioether ordisulfide bond.

According to a particularly advantageous feature, the present inventionrelates to a process for the preparation of an immunotoxin having thestructure II, in which P', W' and GPIR--1a' are as defined above,wherein a protein of the formula:

    P.sub.1' --(Z--Y--E).sub.n --SH                            (IV)

is reacted, in aqueous solution and at room temperature, with a proteinof the statistical formula:

    P.sub.2' --Z'--Y'--E'--G                                   (V)

in which P_(1') and P_(2') represent the radicals of the protein P₁ andP₂ bonded to the groups belonging to the said proteins, or, only if P₁and P₂ are an antibody or antibody fraction, the radicals of theproteins P₁ and P₂ originating from the opening of the carbohydratestructures by reaction with periodic acid, Z, Z', Y, Y', E and E' are asdefined above an G represents a group: ##STR4## or a group --S--S--X, inwhich X is an activating group.

Therefore, both P and GPIR--1a are proteins which arbitrarily have:

(1) the thiol group or groups taking part in the coupling, and

(2) one or more functional groups capable of reacting with the abovethiol groups to form a disulfide or thioether bond.

According to the present invention, the said thiol groups and functionalgroups are those of the native proteins P or GPIR--1a or alternativelyare introduced therein artificially.

Protection of the thiol groups of the starting GPIRs is only necessaryif the said thiol groups are those which are to be used for couplingwith the antibody. If other functional groups are used for the coupling,for example the phenolic hydroxyl of the tyrosines, protection is notcarried out.

Blocking is carried out by reaction with a reagent capable ofsubstituting the SH groups with a radical which can subsequently beremoved by reduction or thiol/disulfide exchange, for example2,2'-dinitro-5,5'-dithiodibenzoic acid (DTNB) or alternatively3-(2-pyridyldisulfanyl) propionic acid. In the absence of such atreatment, the free thiols of the A chain may disappear during thereaction of oxidation and of reduction in the presence of amines, inwhich case they cannot be totally regenerated by reaction with areducing agent such as 2-mercaptoethanol. The excess blocking agent isremoved by dialysis.

The glycoprotein which inactivates ribosomes and the thiols of which areblocked is then subjected to oxidation with periodate ions and toreduction in the presence of amines. If, on the other hand, thecytotoxic sub-unit does not container thiol, or alternatively if thethiol or thiols are not used for coupling, the blocking indicated aboveis not carried out.

The preparation of the conjugates or immunotoxins from theprolonged-action glycoproteins which inactivate ribosomes is carried outby any process suitably chosen from the range of processes described inU.S. Pat. No. 4,340,535. If the chosen cytotoxic sub-unit naturallycontains at least one thiol making it suitable for coupling, this groupwill be preferably used by reaction with the antibody or antibodyfragment carrying an activated disulfide group. If the chosen cytotoxicsub-unit does not naturally possess a thiol group making it suitable forcoupling, at least one functional group carrying a free thiol canpreferably be introduced artificially into the said sub-unit, after thestep of oxidation with periodate ions and reduction in the presence ofan amine, by any known process and the coupling can be continued asindicative above.

The introduction of the said functional group can take place eitherbefore the step of oxidation with periodate ions and reduction in thepresence of an amine, in which case it will be necessary of the thiolradical to be blocked during the step of oxidation and reduction in thepresence of an amine and then unblocked after this step, namely afterthe step of oxidation and reduction in the presence of an amine.

The chemical coupling of the GIPR--1a with the antibody (or antibodyfragment) can be effected by procedures which:

preserve the respective biological activities of the two components ofthe conjugate, namely the antibody and the GPRI--1a,

ensure that the process has a satisfactory reproducibility and a goodcoupling yield,

make it possible to control the value of the ratio GPIR--1a/antibody inthe conjugate obtained, and

lead to a stable and water-soluble product.

Among the procedure corresponding to these characteristics, preferencemust be given to those which involve one or more thiols groups informing the bond between the 2 proteins. In fact, these thiol groups areparticularly suitable for forming either disulfide bonds or thioetherbonds, both of which satisfy the general conditions above.

The preparation of immunotoxins simultaneously having the followingcharacteristics:

the covalent bond between the A chain of ricin and the antibody containsa disfulfide radical,

one of the sulfur atoms forming the disulfide bond is always the sulfuratom belonging to the cysteine residue in the 257-position of the Achain of ricin, and

the link joining the A chain of ricin to the antibody is fixed to thelatter at NH₂ side groups or end groups of a peptide chain.

The coupling of an antibody with the A chain of ricin is described indetail in U.S. Pat. No. 4,340,535.

The same method can be applied to the preparation of immunotoxins havingthe same characteristics and formed by the coupling of an antibody orantibody fragment with a GPIR--1a.

The preparation of immunotoxins formed by the coupling of an antibody orantibody fragment with a GPIR--1a and by a covalent bond of thedisulfide or thioether type at different functional groups is describedin detail below.

In general, in order to carry out the coupling reactions betweenproteins successfully and to eliminate disordered crosslinkings inparticular, it is important for one of the proteins to be coupled, andone only, to carry the thiol or thiol groups to be used, while the otherprotein only carries one or more groups capable of reacting with thethiols in an aqueous medium having a pH of between 5 and 9, and at atemperature not exceeding 30° C., to produce a stable and clearlydefined covalent bond.

The characteristics of the proteins P₁ and P₂ used as starting materialsare illustrated in detail below. The spacing structure E can be replacedwith the preferred structures R to R₈, which are only given as examples.

I--THE PROTEIN P₁

As this protein is in all cases the one carrying the thiol group orgroups which will take part in the coupling, the situation which arisesvaries according to the nature of this protein P₁.

A) In the natural state, the protein P₁ carries one or more thiolradicals which can be used to permit coupling with the protein P₂ ; thisis particularly the case if the protein P₁ is the antibody fragmentknown as F(ab)', as conventionally obtained by limited proteolysis ofthe antibody in the presence of pepsin, followed by reduction of thedisulfide bridge (or bridges) between high-molecular chains.

This is also the case if the protein P₁ as a GPIR--1a, for example themodified A chain of ricin (A-la), or a derivative thereof, in which atleast one of the thiol groups carried by the cysteine 171 and 257residues of the native A chain of ricin is free and accessible forchemical coupling.

In all these cases, the protein P₁ carrying its natural thiol group (orgroups) can be used in this state for the coupling step.

B) In the natural state, the protein P₁ does not carry thiol radicalswhich can be used to permit coupling with the protein P₂ :

this is especially the case if the protein P₁ is a nativeimmunoglobulin, a whole antibody or an antibody fragment, especially oneof the fragments conventionally called F(ab)'₂ or F(ab);

another case in which the protein P₁ does not carry, in the naturalstate, a thiol group which can be used for coupling is the case wherethis protein P₁ is a GPIR--1a, for example the prolonged-action A chainof ricin, in which each of the two cysteine residues is either blockedby alkylation or inaccessible for chemical modification.

In all cases, it will thus be appropriate artificially to introduce intosuch molecules one or more thiol groups capable of permitting coupling.

Three types of reaction can preferably be used for the introduction ofthiol groups:

1--The first type of reaction is with S-acetylmercaptosuccinicanhydride, which is capable of acylating amino groups of the protein. Itwill then be possible to free the thiol groups by reaction withhydroxylamine to remove the acetyl protecting radical, in the manneralready described (Archives of Biochemistry and Biophysics, 119, 41-49,1967). It will even be possible, in the case where the thiol group (orgroups) thus introduced in the protected form are subsequently to reactwith an activated mixed disulfide radical, to dispense with the priordeprotection by means of hydroxylamine; in fact, the reaction creatingthe disulfide bond using the reactants forming the subject of thepresent invention takes place just as well with the S-acetyl radical aswill the free thiol.

Other methods described in the scientific literature can also be used tointroduce thiol groups into the protein to be modified.

2--The second type of reaction consists in reacting the protein via itscarboxyl groups with a symmetrical diamino molecule having a disulfidebridge, of the formula:

    H.sub.2 N--R.sub.1 --S--S--R.sub.1 --NH.sub.2

in which R₁ is an aliphatic group containing from 2 to 5 carbon atoms.

The reaction is preferably carried out with cystamine [R₁ =--(CH₂)₂ --]in the presence of a coupling agent such as a carbodiimide andespecially a water-soluble derivative like1-ethyl-3-dimethylaminopropyl-3-carbodiimide, and leads to theformation, depending on the stoichiometries used, of one of thefollowing derivatives or a mixture of both:

    P.sub.1' --CO--NH--R.sub.1 --S--S--R.sub.1 --NH.sub.2      (Ia)

    P.sub.1' --CO--NH--R.sub.1 --S--S--R.sub.1 --NH--CO--P.sub.1 (Ib).

A reaction product of this type can then be used in two ways:

a) If, in the formulae Ia and Ib, the protein P₁ is a GPIR--1a, forexample the prolonged-action A chain of ricin or one of its derivatives,the reaction medium obtained is subjected, without fractionation, to theaction of a reducing agent such as 2-mercaptoethanol, giving a singleprotein derivative of the general formula:

    P.sub.1' --CONH--R.sub.1 --SH.

The product thus obtained is the purified by dialysis or gel filtration.

b) If, in the formulae Ia and Ib, the protein P₁ is an antibody or oneof its fragments, the reaction medium obtained will be used as such forthe coupling, in which case a thiol/disulfide exchange method will beused, for example the one described by Gilliland and Collier (CancerResearch, 40, 3564, 1980).

3--The third type of reaction consists in using carbohydrate units,which are present in the natural state in the antibodies, in order tofix the radical carrying the thiol which it is proposed to introduce.The protein P is then subjected to oxidation with periodate ions by theknown methods in order to create aldehyde groups on the carbohydrateunits. After the reaction has been stopped by the addition of excessethylene glycol and the by-products and excess reactants have beenremoved by dialysis, the product obtained is treated with a symmetricaldiamino molecule having a disulfide bridge, of the general formula:

    H.sub.2 N--R.sub.1 --S--S--R.sub.1 --NH.sub.2

in which R₁ is n aliphatic group containing from 2 to 5 carbon atoms.The addition products formed are then reduced to secondary or tertiaryamines by reaction with a suitable metal hydride, especially sodiumborohydride. The reaction is preferably carried out with cystamine [R₁=--(CH₂)₂ --] and leads to the formation, depending on thestoichiometries used, of one of the following derivatives or a mixtureof both: ##STR5##

The reaction medium obtained may then be treated exactly as indicatedabove for the products characterized by the structures Ia or Ib.

In the last two types of reaction, described above, for the artificialintroduction of thiol groups (the types using a symmetrical diaminodisulfide reactant), the protein P₁ used preferably possesses neitherfree SH groups nor free amino groups.

In the case of the GPIR--1a, this can always be achieved by alkylationof the natural SH group or groups by reaction with a customary reagentfor thiols, such as N-ethylmaleimide or iodoacetic acid or one of itsderivatives, and by methylation of the natural NH₂ groups in accordancewith the reductive methylation process described in MEANS and FEENEY(Biochemistry 7, 2192 (1968)). For example, up to 6 methyl radicals permol can be introduced beforehand into the modified native A chain ofricin. A protein of this type retains all its biological properties andespecially its capacity to inhibit ribosomal protein synthesis ineucaryotic cells.

In the cases of antibodies or antibody fragments and, more generally,all the substances of the first group, as defined previously, which donot possess naturally free SH groups, it will be appropriate to carryout a reductive methylation, for example by the method of MEANS andFEENEY; in this way, it is usually possible to introduce several dozenmethyl radicals per mol of antibody without modifying its capacity toselectively recognize an antigen on the surface of the cells carryingthis antigen.

II--THE PROTEIN P₂

This protein is in all cases the one which carries one or morefunctional groups capable of reacting with the thiols of the protein P₁to form either a disulfide or a thioether bond. These functional groups,which are always introduced artificially into the protein P₂, differaccording to whether it is desired to effect coupling by a disulfidebond or by a thioether bond and are chosen as indicated below.

THE DISULFIDE BOND

In this case, the preparation of the conjugate can be represented by theequation:

    P.sub.1' --(Z--Y--E).sub.n --SH+P.sub.2' --Z'--Y'--E'--S--S--X→P.sub.1' --(Z--Y--E).sub.n --S--S--E'--Y'--Z'--P.sub.2' +X--SH

The protein P₂ substituted by an activated sulfur atom is obtained fromthe protein P₂ itself or from the correctly protected protein P₂ bysubstitution with a reagent which itself carries an activated sulfuratom, according to the equation:

    P.sub.2 +L--Y'--R--S--S--X→P.sub.2' --Z'--Y'--R'--S--S--X

in which:

P₂ denotes the protein to be substituted and

L--Y' represents a group permitting the covalent fixation of the reagentto the protein.

The functional group L--Y' is a group capable of bonding covalently withany one of the groups carried by the side chains of the constituentamino acids of the protein to be substituted. Among these groups, thefollowing may be singled out in particular:

a) The amino end groups of the peptide chains or the amino side groupsof the lysyl radicals contained in the protein. In this case, L--Y' canrepresent especially:

a carboxyl group which can bond to the amino groups of the protein inthe presence of a coupling agent such as a carbodiimide and especially awater-soluble derivative like1-ethyl-3-dimethylaminopropyl-3-carbodiimide;3-(2-pyridyldisulfanyl)propionic acid activated by the above mentionedcarbodiimide may be used for this purpose.

a carboxylic acid chloride which is capable of reacting directly withthe amino groups to acylate them;

a so-called "activated" ester such as an ortho- or para-nitrophenyl or-dinitrophenyl ester, or alternatively an N-hydroxysuccinimide ester,which can react directly with the amino groups to acylate them, such asthe N-succinimidyl-3-(2-pyridyl-dithio) propionate

an internal anhydride of a dicarboxylic acid, such as, for example,succinic anhydride, which reacts spontaneously with the amine groups tocreate amide bonds; or

an imidoester group: ##STR6## in which R is an alkyl group, which reactswith the amino groups of the protein P₂ according to the equation:##STR7## in which R₃ represents the group --R--S--SX;

b) the phenol groups of the tyrosyl radicals contained in the protein.In this case, L--Y' can represent especially an imidazol-1-ylcarbonylgroup, which reacts with the phenol groups of the protein according tothe equation: ##STR8## in which the imidazol-1-yl is L, the CO group isY' and R₄ is the group --R--S--S--X.

The radical --S--S--X denotes an activated mixed disulfide capable ofreacting with a free thiol radical. In particular, in this mixeddisulfide, X can denote a pyridin-2-yl or pyridin-4-yl group optionallysubstituted by one or more alkyl, halogen or carboxyl radicals. X canalso denote a phenyl group preferably substituted by one or more nitroor carboxyl groups. Alternatively, X can represent an alkoxycarbonylgroup such as the methoxycarbonyl group.

The radical R denotes the spacing structure (indicated as E in thegeneral formula II above) capable of carrying the substituents Y' andS--S--X simultaneously. It must be chosen so as not to contain groupscapable of interfering, during the subsequent reactions, with thereactants used and the products synthesized. In particular, the group Rcan be a group --(CH₂)_(n) --, n being between 1 and 10, oralternatively a group: ##STR9## in which R₆ denotes hydrogen or an alkylgroup having from 1 to 8 carbon atoms and R₅ denotes a substituent whichis inert towards the reactants to be used subsequently, such as acarbamate group; ##STR10## in which R₇ denotes a linear or branchedalkyl group having from 1 to 5 carbon atoms, especially the tert.-butylgroup.

The reaction of the compound L--Y'--R--S--S--X with the protein P₂ iscarried out in a homogeneous liquid phase, most commonly in water or abuffer solution. If necessitated by the solubility of the reactants, awater-miscible organic solvent can be added to the reaction medium at afinal concentration which can reach 20% by volume in the case of atertiary alcohol, such as tertiary butanol, or 10% by volume in the caseof dimethylformamide or tetrahydrofuran.

The reaction is carried out at room temperature for a period of timevarying from a few minutes to a few hours, after which the low molecularweight products, and in particular the excess reactants, can be removedby dialysis or gel filtration. This process usually makes it possible tointroduce between 1 and 15 substituent groups per mol of protein.

When using such compounds, the coupling with the protein P₁ is carriedout by bringing the two proteins together in an aqueous solution havinga pH of between 6 and 8, at a temperature not exceeding 30° C., for aperiod of time varying from 1 hour to 24 hours. The aqueous solutionobtained is dialyzed, if appropriate, to remove the low molecular weightproducts, and the conjugate can then be purified by a variety of knownmethods.

The Thioether Bond

In this case, the preparation of the conjugate consists in reactingP_(1') --(Z--Y--E)_(n) --SH with the protein P₂ into which one or moremaleimide radicals have been introduced beforehand.

The reaction is then represented by the following equation, which isgiven as an example: ##STR11## in which:

R₈ represents an aliphatic or aromatic spacing structure containing from1 to 15 carbon atoms, which is inert towards the reactants to be usedsubsequently, and

Z' represents groups which can vary according to the type of functionalgroup substituted on the protein P₂.

Thus, Z'=oxygen in the case of an ester on the phenol of a tyrosylresidue, Z'=NH in the case of the coupling of an activated carboxylgroup with an amino group of the protein, or Z'=NH--CH₂ in the case ofthe reaction of a chloromethyl ketone with an amino group of theprotein.

The protein P₂ substituted by the maleimide group or groups is obtainedfrom the protein P₂ itself, or the correctly protected protein P₂, bysubstitution of suitable groups of the protein with a reagent whichitself carries the maleimide group. Among these suitable groups, thefollowing may be singled out in particular:

a) The amino end groups of the peptide chains or the amino side groupsof the lysyl residues contained in the protein. In this case, thereagent carrying the maleimide radical can be:

either a reagent of the general formula: ##STR12## in which L--CO--represents:

either a carboxyl group, in which case the reaction is carried out,after activation of the carboxyl group, in the presence of a couplingagent such as a carbodiimide and especially a water-soluble derivativesuch as 1-ethyl-3-dimethylaminopropyl-3-carbodiimide,

or a so-called "activated" ester such as an ortho- or para-nitrophenylor -dinitrophenyl ester, or alternatively an N-hydroxysuccinimide ester,which reacts directly with the amino groups to acylate them.

The preparation of such reagents is described especially in HelveticaChimica Acta 58, 531-541 (1975). Other reagents in the same class arecommercially available.

or a reagent of the general formula: ##STR13## which is capable ofreacting with the amino groups of the protein P₂ according to theequation: ##STR14##

b) the phenol groups of the tyrosyl radicals contained in the protein.In this case, the reagent carrying the maleimide radical can be areagent of the general formula: ##STR15## which reacts with the phenolgroups of the protein according to the equation: ##STR16##

The reaction of the maleimide-carrying reagents with the protein P₂ iscarried out in a homogeneous liquid phase, most commonly in water or abuffer solution. If necessitated by the solubility of the reactants, awater-miscible organic solvent can be added to the reaction medium at afinal concentration which can reach 20% by volume in the case of atertiary alcohol, such as tertiary butanol, or 10% by volume in the caseof dimethylformamide or tetrahydrofuran.

The reaction is carried out at room temperature for a period of timevarying from a few minutes to a few hours, after which the low molecularweight products, and in particular the excess reactants, can be removedby dialysis or gel filtration. This process usually makes it possible tointroduce between 1 and 15 substituent groups per mol of protein.

When using such compounds, the coupling with the protein P₁ is carriedout by bringing the two proteins together in an aqueous solution havinga pH of between 6 and 8, at a temperature not exceeding 30° C., for aperiod of time varying from 1 hour to 24 hours. The solution obtained isdialyzed, if appropriate, to remove the low molecular weight products,and the conjugate can then be purified by a variety of known methods.

The compounds of the formula: ##STR17## in which E and G are as definedabove, are prepared by a process which comprises reacting a compound ofthe formula:

    G--E--COOH                                                 VII

in which G and E are as defined above, with the carbonyldiimidazole ofthe formula: ##STR18## in an organic solvent at a temperature of 10° to40° C.

The compounds of the formula VI are particularly useful as agents forcoupling with the hydroxyls of the tyrosines of the proteins GPIR--1aand P.

According to another feature, the present invention relates to newproducts having the following statistical formula:

    GPIR--1a"--O--CO--E--G                                     IX

in which:

GPIR--1a" represents the radical of the protein GPIR--1a or any moleculederived from the said GPIR--1a by artificial modification of any one ofits functional groups, from which one or more phenolic hydroxyl groupsof the tyrosines have been removed;

the oxygen atom is that belonging to the phenolic hydroxyl groupsmissing from the radical GPIR--1a"; and

E and G are as defined above.

Particular preference is given to the compounds of the formula IX inwhich E represents a group --(CH₂)_(p) --, in which p is an integer from2 to 7, or a group: ##STR19## and G is a group of the structure--S--S--X, in which X is an activating radical chosen from thepyridin-2-yl and pyridin-4-yl groups which are unsubstituted orsubstituted by one or more halogens or alkyl, carboxyl or alkoxycarbonylradicals, the phenyl group which is unsubstituted or substituted by oneor more halogens or nitro, alkoxy, carboxyl or alkoxycarbonyl groups, oran alkoxycarbonyl group.

The products of the formula IX are prepared by reacting a product of theformula:

    GPIR--1a"--OH

in which GPIR--1a" is as defined above and the hydroxyl group is thephenolic hydroxyl missing from the tyrosines of the radical GPIR--1a",with a compound of the formula VI above, at a temperature of 10 to 40°C., in an aqueous solvent optionally containing a water-miscible organicsolvent such as, for example, an ether solvent like dioxane ortetrahydrofuran.

In the case where GPIR--1a is the prolonged-action a chain of ricin, theproperties of the resulting immunotoxins IT (A--1a) are as follows:

the average degree of coupling, expressed as the number of mol ofmodified A chain per mol of antibody, is usually between 0.5 and 5 andin particular between 1 and 3,

the separation of the IT (A--1a) by polyacrylamide gel electrophoresisresults in a splitting of the product into a series of bandscorresponding to products whose molecular weights differ from that ofthe antibody by successive increments of 30,000 daltons,

the studies performed by cytofluorometry make it possible to show thatthe antibody has not undergone any substantial degradation during theactivation and coupling reactions to which it has been subjected, andthat it is still capable, within the conjugate itself, of recognizingthe antigen against which it is directed, and

the inhibitory activity of the A chain, modified and coupled with anantibody, on protein synthesis, determined in an acellular model in thepresence of 2-mercaptoethanol, is totally retained.

The cytotoxic activity of the immunotoxins IT (A--1a), measured in atest for protein synthesis in a cell model on the cells having thetarget antigen, is more than 100 times greater than that measured underthe same conditions on cells not having the target antigen. For example,the immunotoxin (denoted by IT (A--1a) AT15E) built up by coupling theA--1a chain of ricin, by means of a link containing a disulfide bridge,with a monoclonal antibody (denoted by antibody AT15E) directed againstthe antigen Thy 1.2 present on the surface of certain mice leukemiacells is about 1000 times more cytotoxic towards the positive Thy.1 2cells than towards the negative Thy 1.2.

Finally, after intravenous administration of IT (A--1a) to rabbits at adose of the order of 0.4 l mg/kg of body weight, expressed as A chain,the plasma level of IT (A--1a) present in the bloodstream 24 hours afterinjection is 10 to 200 times greater than the plasma level of theconventional IT measured under the same conditions. Thus, in a typicalcase involving rabbits, it is found that the plasma level of IT (A--1a)AT15E in the bloodstream 24 hours after injection is 10% of the productpresent at time zero, as against 0.08% for the correspondingconventional IT AT15E after the same time, i.e. an increase by a factorof the order of 120.

This gives modified immunotoxins which have acquired a new character asregards their pharmacokinetic properties.

More particularly, by appropriate modification of the cytotoxicsub-unit, it has been possible to add to the specific cytotoxicityproperties of immunotoxins, without interfering with them, a newproperty which is just as intrinsic, namely the capacity to show slowplasma elimination kinetics after injection to superior animals orhumans.

The invention also relates to the anticancer pharmaceutical compositionscontaining, as active principle, an immunotoxin modified according tothe invention, in combination with a pharmaceutically acceptablevehicle, suitable for administration by injection, and in particular byintravenous injection.

The following examples are given non-restrictively to illustrate theinvention.

EXAMPLE 1

This example demonstrates the slow elimination of the A chain of ricinmodified by oxidation reaction with sodium periodate, in the presence ofan excess of L-leucine, after intravenous injection into the animal.

I--Modification of the A chain of ricin by the simultaneous action ofsodium periodate and L-leucine

1) Blocking of the natural SH of the A chain with DTNB

The A chain of ricin was prepared and purified in the manner indicatedin U.S. Pat. No. 4,340,535.

20 equivalents of a solution of 2,2'-dinitro-5,5'-dithiodibenzoic acid(DTNB), i.e., 1 ml of a solution of DTNB in a 125 mM phosphate buffer,(this solution is brought to pH 7 with sodium hydroxide), are added to23 ml of a solution of A chain of ricin (containing 0.84 thiol group perA chain) at a concentration of 7.2 mg/ml in the PBS buffer (a buffer 20mM in respect of phosphate and 150 mM in respect of NaCl, of pH 7). Thesolution is then dialyzed against PBS buffer at 4° C. to give 162 mg ofA chain blocked on the thiol group, as a solution containing 6.9 mg/mlof said A chain.

2) Periodate oxidation and formation of Schiff's base with L-leucine onthe A chain blocked on the thiol function.

12 ml of a solution containing 52 mg/ml of L-leucine in a 30 mMphosphate buffer, pH 6.5 and then 0.5 ml of an aqueous solution of 0.5Msodium periodate are added to 79.4 mg of A chain blocked on the thiolfunction contained in 11.5 ml of solution brought to pH 6.5 with aceticacid. Incubation is left to proceed for 17 hours at 4° C. in the dark.The reaction is stopped by the addition of 2.8 ml of a 1M aqueoussolution of ethylene glycol. After incubation for 15 minutes at 20° C.,the reaction medium is dialyzed at 4° C. against PBS buffer for 48hours. After centrifugation at 10,000×g for 30 minutes, 28 ml of asolution of A chain blocked on the thiol function and modified on itsosidic residues, by oxidation and formation of a Schiff's base, areobtained at a concentration of 1.9 mg/ml.

3) Unblocking of the thiol groups

25 ml of 1.9 mg/ml solution of A chain blocked and modified on itsosidic residues are brought to 10.5 ml by concentration, and 0.241 ml of50% 2-mercaptoethanol are added. Incubation is left to proceed for 1hour at 20° C. The solution is then dialyzed against PBS buffer at 4° C.This gives 26.5 mg/ml.

Using the DTNB technique (Methods in Enzymology, 1972, 25, 457 (AcademicPress)), it is determined that the modified A chain obtained has 0.89free thiol group per mol. The molecular weight of the modified A chainis 30,000±3,000, determined by polyacrylamide gradient electrophoresisin the presence of sodium dodecylsulfate.

The previously obtained preparation of A chain in which the osidic unitshave been modified was studied for its enzymatic activities in theinhibition of protein synthesis and for its pharmacokinetic properties.

II--Enzymatic activity of the prolonged-action A chain, measured on anacellular model

The fundamental biological property of the A chain of ricin is toinhibit protein synthesis in cells by degradation of the ribosomalsub-unit 60S.

The in vitro protocol involves the use of appropriately complemented,subcellular fractions of rat liver capable of incorporating ¹⁴C-phenylalanine in the presence of an artificial messenger RNA:polyuridylic acid.

The procedure employed for preparing the subcellular fractions andmeasuring the incorporation of ¹⁴ C-phenylalanine is a n adaptation ofthe method described in Biochemica Biophysica Acta 1973, 312, 608-615,using both a microsomal fraction and a cytosol fraction of the rathepatocytes. The sample containing the A chain is introduced in the formof a solution appropriately diluted in a 50 mM Tris HCl buffer of pH 7.6containing 0.2% of 2-mercaptoethanol and 15 micrograms/ml of bovineserum albumin.

The count data are used to calculate, relative to a control mediumwithout inhibitor, the percentage inhibition of the incorporation of ¹⁴C-phenylalanine into the proteins for each reaction medium containing Achain of ricin.

With the curves obtained, it is possible to calculate the IC₅₀ ofconcentration of A chain (native or modified) which inhibits theincorporation of the radiomarked precursor into the proteins. An IC₅₀equal to 3.10-¹⁰ mole/l is thus observed for the modified blocked A-1achain. The IC₅₀ of the control chain in the experiment is of 10-¹⁰mole/l: considering the precision of the measurements, it is clear thatthe modification entails no significant loss of activity for the Achain.

III--Pharmacokinetic properties of the prolonged-action A chain modifiedon its osidic units (A--1a chain)

The native or modified A chain is administered to rabbits by means of asingle injection into a vein in the ear. The quantity of A chaininjected corresponds to 0.415 mg/kg. Blood samples are taken atintervals on heparin. The plasmas are analyzed with the aid of aradioimmunometric test designated below by the abbreviation RIM-1.

This technique has the advantage of determining the A chain withoutmodifying it. This determination is carried out in microtitration plates(for example: "NUNC-TSP screening system" from Poly Labo Block France),the lid of which carries hyperabsorbent spikes which dip into the wellsin the base. These spikes constitute the solid phases. Sheep antibodiesdirected against the A chain of ricin (designated below by theabbrevation Ac1), purified by affinity chromatography, are absorbed onthe solid phases. For this purpose, 200 microliters of a solution of Ac1containing 10 micrograms/ml in PBS phosphate buffer are divided up intothe wells. The spikes are brought into contact firstly with the solutionof Ac1 for 24 h at 4° C. and then with fetal calf serum for 3 h at 20°C. in order to saturate all the fixation sites. The saturatedimmunoabsorbent is then brought into contact for 3 h at 20° C. with theplasma samples to be determined at different dilutions, or withsolutions of A chain of known concentrations in order to establish thecalibration curve. After washing with a PBS buffer, the immunoabsorbentis brought into contact for 2 h at 20° C. with the sheep antibodiesdirected against the A chain of ricin, which have been purified byaffinity chromatography and radiolabeled (designated below by theabbreviation Ac2). The radiolabeling of the Ac2 is effected with iodine125 in the presence of chloramine T by the method of Greenwood andHunter (Biochem. J., 1963, 89, 114); the specific activity of theradiolabeled Ac2 antibodies is 5 to 10 microcuries/microgram. 10⁶ cpm ofradiolabeled Ac2 antibodies are introduced as 200 microliters into a PBSbuffer containing 0.1% of bovine serum albumin. After washing in PBSbuffer, the spikes are detached and the quantity of bound Ac2 ismeasured by counting the radioactivity. The concentration of A chain inthe samples to be determined is measured by reference to the calibrationcurve established by introducing the A chain at different knownconcentrations. When prolonged-action A chain is injected into theanimal, this same prolonged-action A chain is used to establish thecorresponding calibration curve.

The values of the concentration of A chain in the blood plasma measuredby this technique are reproducible and reliable. The detection thresholdis 1 nanogram/ml. A study of the reproducibility within and betweenexperiments gives coefficients of variation of less than 10% forconcentration values within the range from 1 to 200 nanograms/ml.

The results of these experiments are represented in the form of curvesin which the time, expressed in hours, is plotted on the abscissa andthe plasma concentration of the product measured, recorded in percent ofthe theoretical plasma concentration at time zero, is plotted on alogarithmic scale on the ordinate. This value, called the "relativeplasma concentration" (RPC), is calculated using the followingexpression: ##EQU1##

The plasma volume is considered to be equal to 36 ml/kg of the animal'sbody weight.

FIG. 1 shows the plasma elimination curve, as a function of time, forthe native A chain of ricin injected intravenously. This curve (curve 1)has two phases: in the first phase, the product disappears very rapidlyfrom the bloodstream since only 0.1% of the dose administered remains inthe plasma three hours after injection. In the second phase, thedecrease is slower.

When the A chain has been modified on its osidic units, (A-1a chain,curve 2) the elimination profile is profoundly modified: the firstelimination phase--which is responsible for the disappearance of themajority of the product--is practically suppressed, which leads to aconsiderable increase in the plasma levels of A chain. Twenty hoursafter injection, the concentration of the oxidized A chain is 460 timesgreater than in the case of the unmodified A chain (curve 2).

These results .;then prove that the oxidation reaction with sodiumperiodate and the blocking by formation of Schiff's base from L-leucinehave modified the sugars involved in the recognition process responsiblefor the elimination of the A chain, to the point of preventing thatrecognition without the characteristic biological activity of the Achain being altered.

EXAMPLE 2

This example shows, after intravenous injection into the animal, theslow elimination of the A chain of ricin modified by oxidation reactionwith sodium periodate and by formation of Schiff's base in th e presenceof L-alanine, or L-glutamic acid or L-arginine.

A) Procedure followed

The A chain is modified according to the method described in Example 1,except that the Schiff's reaction is carried out in the presence eitherof glutamic acid, or arginine, or alanine, instead of leucine. Theintroduced quantities of each of these amino acids are 10³, 5.10³, and10⁴ times greater than the molar quantities of A chain used, for theglutamic acid, the arginine and the alanine, respectively.

B) Results

Table I shows the properties of these different modified A--la chains.The results obtained with native A chain and with modified A--1a chainby sodium periodate and L-leucine such as described in Example 1 arerepeated for comparative purposes.

                  TABLE I                                                         ______________________________________                                                       Primary amines used for the                                                   formation of Schiff's bases                                               Native        Glu-                                                            A     L-      tamic   L-arg-                                                                              L-                                                chain leucine acid    ginine                                                                              alanine                                ______________________________________                                        Inhibition of protein                                                                      10      3       3.4   2     3.3                                  synthesis in                                                                  acellular model                                                               (IC.sub.50 × 10-.sup.10 M)                                              Free thiol per A chain                                                                     0.9     0.89    0.87  0.8   0.85                                 Molecular weight                                                                           30,000  30,000  30,000                                                                              30,000                                                                              30,000                               (±3,000)                                                                   Relative plasma                                                                            0.01    7       7     8.5   10                                   concentration (%)                                                             24 h after                                                                    injection                                                                     ______________________________________                                    

These results prove that the oxidation reactions with sodium periodatetogether with the simultaneous blocking of the aldehyde groups in theform of Schiff's bases by the L-alanine, L-arginine or L-glutamic acidhave modified the sugars involved in the recognition process responsiblefor the biological elimination of the A chain in vivo to the point ofpreventing the main part of that recognition, without the ribosomesinactivation property of the A chain being significantly altered andwithout any loss of the thiol groups which can be used for subsequentcouplings.

EXAMPLE 3

The immunotoxin (abbreviated IT) obtained between an antibody directedagainst mice T cells (antibody directed against antigen Thy 1.2)substituted by activated, disulfide groups and the A--1a chain of ricin

a) Antibody directed against mice T cells or AT 15E antibody;

This antibody was obtained according to the method described in Journalof Immunology, 1979, 122, 2491-2498.

b) A--1a chain of ricin:

The A--1a chain of ricin which was used was prepared as indicated inExample 1.

c) Activated antibody directed against mice T cells

3.62 mg of N-succinimidyl-3-(2-pyridyl-dithio)propionate in ethanol at95% under a volume of 0.326 ml, are added to 43.7 ml of a 7 mg/mlsolution of antibody in a phosphate buffer 125 mM of pH 7. The mixtureis stirred for 30 minutes at 20° C. After dialysis against a 125 mMphosphate buffer of pH 7, the protein solution is centrifuged at10,000×g for 30 minutes at 4° C., and 96.8 mg of activated antibody arethus obtained at a concentration of 2.42 mg/ml. By spectrophotometricdosage at 343 nm of 2-pyridinethione liberated by exchange with2-mercaptoethanol, it is found that an antibody is obtained whichcarries 6.7 activated mixed disulfide groups per mole of antibody.

d) Preparation of the immunotoxin having the prolonged-action A--1achain of ricin.

10 ml of A--1a chain at 2.65 mg/ml obtained as indicated in Example 1are added to 14.8 ml of the activated antibody solution obtainedhereinabove (concentration 2.42 mg/ml., i.e. 35.8 mg of activatedantibodies), and incubation is left to proceed for 20 hours at 25° C.The solution is centrifugated, then purified by filtration on a gel (AcA44 gel) with measurement of the optical density of the effluent at 280nm. Regrouping of the fractions containing both the antibody and themodified A chain leads to 52 ml of immunotoxin solution at 0.315 mg/ml,i.e. 18.25 mg. This solution contains 0.076 mg of modified A--1a chaincoupled to the antibody per ml. The average coupling rate of thispreparation is therefore 1.6 mole of A--1a chain per mole of antibody.

The immunotoxin with A--1a chain of ricin obtained as indicated abovewas studied for its pharmacokinetic properties and its specificcytotoxic properties towards the target cells.

EXMAPLE 4

This example shows how the slow plasma elimination property is acquiredby the prolonged-action immunotoxins with A chain of recin, abbreviatedto IT (A---1a).

A) Procedure

The conjugate prepared according to the method describe din Example 3 isadministered to the rabbit by a single injection in a vein of the ear.The injected quantity corresponds to 0.415 mg/kg expressed in A chain.Blood samples are taken at intervals on heparin. The plasmas areanalyzed with the aid of radioimmunometric tests with two siteshereinafter designated by the abbreviation RIM-3.

This assay is carried out by the same technique as that used for thetest RIM-1 (described in example 1) except that the solution Ac2 is herea solution of goat antibodies directed against mouse IgG, purified byaffinity chromatography and radiolabeled as described for the RIM-1technique (described in Example 1). The concentration of modifiedimmunotoxin in the samples is measured by reference to a calibrationcurve established by introducing the modified immunotoxin at differentknown concentrations. The assay RIM-3 has the same reliability andreproducibility characteristics as described for the RIM-1 technique.

By way of comparison, a control study is carried out under the sameconditions with the conjugate called IT-AT15E, which is obtained by thereaction of the same antibody AT15E, substituted by activated disulfidegroups, with the native A chain of ricin. The preparation of thisconjugate is carried out by the same method as described in Example 3.The results of these pharmacokinetic experiments are represented in thesame way as for the uncoupled A chain of ricin in Example 1.

b) Results

FIG. 2 shows the plasma elimination curves, as a function of time, forIT AT15E (curve 1) and IT (A--1a) AT15E (curve 2), injectedintravenously, 24 hours after injection, the concentration of activeimmunotoxin is 120 times greater for IT (A--1a) AT15E than for theconventional ITAT15E. This fact demonstrates that the newpharmacokinetic properties of the oxidized A chain are retained aftercoupling with an antibody.

EXAMPLE 5

This example demonstrates the retention of the specific cytotoxicityproperties of IT (A--1a) AT15E towards the positive target cells Thy1.2.

The fundamental biological property of the A chain of ricin is toinhibit protein synthesis in cells by degradation of the ribosomalsub-unit 60S. The technique uses a cell model in which the effect of thesubstances studied on the incorporation of ¹⁴ C-leucine into cancerouscells in culture is measured.

The cells used belong to the cell line T2 derived from a T leukemiawhich carries the antigen Thy 1.2. The cells are incubated in thepresence of the substance to be studied, and then, when incubation hasended, the degree of incorporation of ¹⁴ C-leucine by the cells treatedin this way is measured.

This measurement is made by a technique adapted from the one describedin Journal of Biological Chemistry 1974, 249(11), 3557-3562, using thetracer ¹⁴ -C leucine to determine the degree of protein synthesis. Theradioactivity incorporated is determined here on the whole cellsisolated by filtration.

On the basis of these determinations, it is possible to draw thedose/effect curves, plotting, on the abscissa, the molar concentrationof A chain in the substances studied, and, on the ordinate, theincorporation of ¹⁴ C-leucine expressed as a percentage of theincorporation by control cells in the absence of any substance affectingprotein synthesis.

It is thus possible to determine, for each substance studied, theconcentration which causes a 50% inhibition of the incorporation of ¹⁴C-leucine, or "50% inhibitory concentration " (IC₅₀).

Table II hereunder shows the values of IC₅₀ obtained in the sameexperiment with IT-(A-1a)-AT15E and IT At15E, on the one hand, thenative A chain and the uncoupled A-1a chain on the other hand.

It is found that the IT(A-1a)AT15E has a very strong cytotoxic activity,identical to that obtained with the corresponding immunotoxin containingthe native A chain and which is about 1000 times greater than that ofthe uncoupled modified A-1a chain, measured in the same conditions.

                  TABLE II                                                        ______________________________________                                        Tested Product  IC.sub.50                                                     ______________________________________                                        IT (A-1a) AT15E 2.10-.sup.10 M                                                IT AT15E        2.10-.sup.10 M                                                native A chain  3.10-.sup.7 M.sup.                                            A-1a chain      2.10-.sup.7 M.sup.                                            ______________________________________                                    

EXAMPLE 6

This example shows, after intravenous injection into the animal,

1) the rapid elimination of native gelonine and

(2) the slow elimination of gelonine modified by oxidation reaction withsodium periodate in the presence of L-leucine.

A) Modification of gelonine by the simultaneous action of sodiumperiodate and L-leucine.

The gelonine was prepared and purified from Gelonium multiflorum by themethod which has been described (J. Biol. Chem. (1980) 255, 6947-6953).The modification reaction is carried out under the same conditions asthose described for the A chain of ricin in Example 1, except that thestep in which the thiols are blocked with DTNB is omitted.

In fact, as the coupling if gelonine with the antibody is not generallyperformed using natural thiol groups of the gelonine, the thiol groupswill be introduced artificially, after the oxidation step, by thetechnique described in Cancer Res., 1984, 44, 129-133.

1 ml of a 52 mg/ml solution of L-lecuine in a 30 mM phosphate buffer ofpH 6.5, followed by 40 microliters of a 0.5M solution of sodiumperiodate in water are added to 1 ml of a solution containing 3 mg/ml ofgelonine in PBS buffer, brought to pH 6.5 with 1M acetic acid.Incubation is left to proceed for 17 hours at 4° C. in the dark. Thereaction is stopped by the addition of 210 microliters of a 1M aqueoussolution of ethylene glycol. After incubation for 15 minutes at 20° C.,the reaction medium is dialyzed at 4° C. against PBS buffer. Aftercentrifugation at 10,000×g for 30 minutes, this gives 2.9 mg of oxidizedgelonine at a concentration of 2.5 mg/ml.

Like the A chain of ricin, the fundamental property of gelonine is toinhibit protein synthesis in eucaryotic cells by degradation of theribosomal sub-unit 60S (Biochem. J., 1982, 207, 505-509). In the case ofgelonine too, the modification does not cause any significant loss ofactivity.

B) --Pharmacokinetic properties of prolonged-action gelonine

Native gelonine or gelonine modified by the procedures explained aboveis administered to rabbits by a single injection into a vein in the ear.The quantity of geloninine injected is between 0.3 and 0.4 mg/kg. Bloodsamples are taken at intervals on heparin. The plasmas are analyzed withthe aid of a radioimmunometric test designated below by the abbreviationRIM-2.

This test is performed by the same technique as used for the test RIM-1,except that the solution Ac1 here is a solution of anti-gelonine rabbitantibodies purified by affinity chromatography, the Ac2 antibodies beingthe same antibodies radiolabeled. The radio-labeling procedure isidentical to that described for the technique RIM-1. The concentrationof native gelonine or modified gelonine in the samples to be determinedis measured by reference to a calibration curve established byintroducing native or modified gelonine at different knownconcentrations. The test RIM-2 has the same reliability andreproducibility characteristics as described for the technique RIM-1.

The plasma elimination curves, as a function of time, for nativegelonine and modified gelonine, injected intravenously show that, thenative gelonine, like the native A chain of ricin, disappears veryrapidly from the bloodstream since 99.99% of the gelonine present in thebloodstream disappears in 24 hours. When the gelonine has been modifiedon its polysaccharide units, the elimination profile is profoundlymodified: 24 hours after injection, the concentration of the modifiedgelonine is 300 times greater than that of the native gelonine.

Thus, as for the A chain of ricin, these results prove that the sodiumperiodiate oxidation reaction and the blocking reaction by formation ofSchiff's bases due to L-lecuine, have modified the sugars involved inthe recognition process responsible for the elimination of the gelonine,to the point of preventing this recognition.

These modified immunotoxins can be used for the treatment of cancerousor non-cancerous diseases where the target cells would be recognized bythe antibody used for preparing the immunotoxin. The optimumadministration conditions, and the treatment time will have to bedetermined in each case according to the subject and to the nature ofthe disease to be treated. The new drugs according to the invention arepresented in a form suitable for administration by injection andpreferably intravenous injection.

What is claimed is:
 1. A modified glycoprotein which inactivatesribosomes and having the ribosome-inhibiting activity of thecorresponding native glycoprotein which inactivates ribosomes and anin-vivo prolonged action with respect to the corresponding nativeglycoprotein which inactivates ribosomes, wherein said modifiedglycoprotein which inactivates ribosomes is obtained by treatment of aglycoprotein which inactivates ribosomes having a molecular weight fromabout 20,000 to 30,000 with an oxidizing agent to oxidize the osidicgroups of said glycoprotein to aldehyde groups which are blocked whensaid aldehdye groups are formed by a primary amine other than saidglycoprotein.
 2. An immunotoxin having a prolonged action in-vivo,resulting from the coupling of an antibody or antibody fragment with amodified glycoprotein which inactivates ribosomes according to claim 1.3. An immunotoxin of claim 2 having the following statistical formula:

    P'--W--GPIR--Ia'

in which P' represents the radical of a protein P, which is an antibodyor an antibody fragment, GPIR--Ia' represents the radical of a proteinGPIR--Ia, which is the glycoprotein which inactivates ribosomes, thecarbohydrate units of which are modified by oxidation, and W representsa divalent covalent structure containing a thioether group or adisulfide group in which either the sulfur atoms are those of thecysteines of P and GPIR--Ia or they are bonded to the groups belongingto P and/or GPIR--Ia by spacing structures carrying a functional groupbonded to the said groups belong to P and/or GPIR--Ia.
 4. Theimmunotoxin of claim 3 having the following statistical formula:

    P'--W--GPIR--Ia'

in which P' represents the radical of a protein P, which is an antibodyor an antibody fragment, GPIR--Ia' represents the radical of a proteinwhich is a glycoprotein which inactivates ribosomes, GPIR--Ia, obtainedby treatment of a glycoprotein which inactivates ribosomes, with anaqueous solution of an alkali metal periodate, for a period of 0.2 to 24hours, at room temperature and in the absence of light, and W representsa divalent covalent structure containing a thioether group or adisulfide group in which the sulfur atoms are either those of thecysteines of P and GPIR--Ia or they are bonded to the groups belongingto P and/or GPIR--Ia by spacing structures carrying a functional groupbonded to the said groups belonging to P and/or GPIR--Ia.
 5. Theimmunotoxin of claim 3 in which W is W' which represents a divalentstructure chosen from:(a) a group of the formula ##STR20## (b) a groupof the formula: ##STR21## (c) a group of the formula:

    --Z--Y--E--S--S--(E'--Y'--Z').sub.n --

or (d) a group of the formula:

    --(Z'--Y'--E').sub.n --S--S--E--Y--Z--,

in which: Z and Z', are the same or different and represent the groupsbelonging to the proteins GPIR--Ia and P, chosen from the oxygen atomoriginating from the hydroxyl of one of the tyrosine residues, thecarbonyl group originating from one of the terminal carboxyls or one ofthe free carboxyls of the aspartic and/or glutamic acids of GPIR--Ia andP, the --NH-- group originating from one of the terminal amines ofGPIR--Ia and P or from one of the amines in the epsilon position of oneof the lysine residues, and, only for Z in the covalent structures (b)and (c), the group originating from the dialdehyde structure obtainedafter oxidation of one of the carbohydrate structures of P with periodicacid; Y and Y' represent functional groups capable of bonding covalentlywith any one of the groups Z and Z' of the proteins GPIR--Ia and P; Eand E' represent inert spacing structures; and n represents zero or 1.6. The immunotoxin of claim 5 having the following statistical formula:

    P'(W'--A--Ia').sub.m

in which m varies from 0.3 to 12, P' represents the radical of a proteinP, which is an antibody or an antibody fragment, GPIR--Ia' is A--Ia'which represents the radical of a glycoprotein which inactivatesribosomes, obtained by treatment of the A chain of ricin, in which atleast one of the thiol groups of its cysteines 171 and 257 is protected,with an aqueous solution of an alkali metal periodate, for a period of0.2 to 24 hours, at a temperature of 0° to 15° C. and in the absence oflight, and by deprotection of the thiol group or thiol groups.
 7. Animmunotoxin as claimed in claim 6 of the statistical formula:

    P'(W'--A--Ia').sub.m

in which W' and A--Ia' are as defined in claim 6, P' is an antibodyfragment Fab or Fab' and m varies from 0.3 to
 2. 8. An immunotoxin asclaimed in claim 6, of the statistical formula:

    P'(W'--A--Ia').sub.m

in which W' and A--Ia' are as defined in claim 6, P' is an antibodyfragment F(ab')₂ and m varies from 0.5 to
 4. 9. An immunotoxin asclaimed in claim 6, of the statistical formula:

    P'(W'--A--Ia').sub.m

in which W' and A-Ia' are as defined in claim 6, P' is an antibody ofthe IgG type and m varies from 0.5 to
 6. 10. An immunotoxin as claimedin claim 6, of the statistical formula:

    P'(W'--A--Ia').sub.m

in which W' and A--Ia' are as defined in claim 6, P' is an antibody ofthe IgM type and m varies from 1 to 12.